Aqueous radiation protecting formulations and methods for making and using them

ABSTRACT

Medical devices are typically sterilized in processes used to manufacture such products and their sterilization by exposure to radiation is a common practice. Radiation has a number of advantages over other sterilization processes including a high penetrating ability, relatively low chemical reactivity, and instantaneous effects without the need to control temperature, pressure, vacuum, or humidity. Unfortunately, radiation sterilization can compromise the function of certain components of medical devices. For example, radiation sterilization can lead to loss of protein activity and/or lead to bleaching of various dye compounds. Embodiments of the invention provide methods and materials that can be used to protect medical devices from unwanted effects of radiation sterilization.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication No. 61/561,146, filed Nov. 17, 2011, the entire contents ofwhich are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to medical devices useful in in vivoenvironments, in particular, methods and materials used to sterilizesuch devices prior to their implantation in vivo.

2. Description of Related Art

Medical personnel and patients commonly utilize a wide variety ofpre-sterilized medical products, such as glucose sensors that are usedby diabetic patients. In this context, a number of differentsterilization processes are used with various medical products in orderto kill microorganisms that may be present. Most sterilization processesrequire the sterilizing agent to systemically permeate the article beingsterilized. These methods can include heat sterilization, where theobject to be sterilized is subjected to heat and pressure, such as in anautoclave. The heat and pressure penetrates though the object beingsterilized and after a sufficient time will kill the harmfulmicroorganisms. Gases such as hydrogen peroxide or ethylene oxide arealso used to sterilize objects. Sterilization methods also include thosethat use ionizing radiation, such as gamma-rays, x-rays, or energeticelectrons to kill microorganisms.

Radiation has a number of advantages over other sterilization processesincluding a high penetrating ability, relatively low chemicalreactivity, and instantaneous effects without the need to controltemperature, pressure, vacuum, or humidity. Consequently, thesterilization of medical devices by exposure to radiation is a commonpractice. Medical devices composed in whole or in part of polymers aretypically sterilized by various kinds of radiation, including, but notlimited to, electron beam (e-beam), gamma ray, ultraviolet, infra-red,ion beam, and x-ray sterilization.

Electron-beam and gamma ray sterilization processes provide forms ofradiation commonly used to kill microbial organisms on medical devices.However, when used to kill microorganisms, such radiation can alter thestructure of functional macromolecules present in medical productsincluding polymers such as proteins. High-energy radiation tends toproduce ionization and excitation in polymer molecules, as well as freeradicals. These energy-rich species can react with macromoleculespresent in medical products and undergo dissociation, abstraction, chainscission and cross-linking.

The deterioration of the performance of polymeric materials and othermacromolecules in medical devices due to radiation sterilization hasbeen associated with free radical formation during radiation exposure.Electron-beam and gamma ray radiation can therefore be problematicalwhen used to sterilize medical device includes components that areradiation sensitive. This complicates the sterilization process andlimits the range of designs or materials available for medical devices.Consequently, methods and formulations that can protect medical devicematerials from damage that can occur as a result of exposure tohigh-energy radiation are desirable.

SUMMARY OF THE INVENTION

As noted above, the sterilization of medical devices by exposure toradiation is a common practice. Unfortunately, radiation sterilizationcan compromise the function of certain components of some medicaldevices. In this context, embodiments of the invention provide methodsand materials that can be used to protect medical devices from unwantedeffects of radiation sterilization. While typical embodiments of theinvention pertain to glucose sensors, the systems, methods and materialsdisclosed herein can be adapted for use with a wide variety of medicaldevices.

The invention disclosed herein has a number of embodiments. Typicalembodiments of the invention comprise methods for inhibiting damage to asaccharide sensor that can result from a radiation sterilization process(e.g. electron beam irradiation) by combining the saccharide sensor withan aqueous radioprotectant formulation during the sterilization process.In common embodiments of the invention, the saccharide sensor comprisesa saccharide binding polypeptide having a carbohydrate recognitiondomain and the aqueous radioprotectant formulation comprises asaccharide selected for its ability to bind the saccharide bindingpolypeptide. In certain embodiments of the invention, the saccharidesensor comprises a fluorophore; and the aqueous radioprotectantformulation comprises a fluorophore quenching composition selected forits ability to quench the fluorophore. In illustrative embodiments ofthe invention, the sensor is a glucose sensor and the saccharide bindingpolypeptide comprises mannan binding lectin, concanavalin A,glucose-galactose binding protein, or glucose oxidase. In certainmethods of the invention, the sterilization process is performed underconditions selected so that the saccharide binds the saccharide bindingpolypeptide and/or the fluorophore quenching composition quenches thefluorophore in a manner that inhibits damage to the saccharide sensorthat can result from the radiation sterilization process.

As discussed below, a number of compounds are useful in theradioprotectant formulations disclosed herein. In certain embodiments ofthe invention, the aqueous radioprotectant formulation comprises asaccharide such as glucose, mannose, fructose, melizitose,N-acetyl-D-glucosamine, sucrose or trehalose. In some embodiments, theaqueous radioprotectant formulation comprises an antioxidant selectedfrom the group consisting of ascorbate, urate, nitrite, vitamin E,α-tocopherol or nicotinate methylester. In certain embodiment of theinvention, the aqueous radioprotectant formulation comprises a bufferingagent, for example, one selected from the group consisting of citrate,tris(hydroxymethyl)aminomethane (TRIS) and tartrate. In variousembodiments of the invention the radioprotectant formulations cancomprise additional agents such as surfactants, amino acids,pharmaceutically acceptable salts and the like. Related embodiments ofthe invention include compositions of matter comprising a medical devicecombined with an aqueous radioprotective formulation. One illustrativeembodiment of the invention is a composition of matter comprising asaccharide sensor that includes a saccharide binding polypeptide; and/ora fluorophore. In typical composition embodiments, a saccharide sensoris combined with an aqueous radioprotectant formulation comprising asaccharide, wherein the saccharide binds to the saccharide bindingpolypeptide. Optionally in such compositions, the saccharide sensor iscombined with a fluorophore quenching compound in the aqueousradioprotective formulation.

A number of compounds can be combined with the saccharide sensorsdisclosed herein to form the radioprotectant compositions of theinvention. In typical embodiments of the invention, the compositioncomprises a saccharide selected from the group consisting of glucose,mannose, fructose, melizitose, N-acetyl-D-glucosamine, sucrose ortrehalose. In certain embodiment of the invention, the compositioncomprises a fluorophore quenching compound, for example, acetaminophen.In some embodiments of the invention, the composition comprises anantioxidant compound is selected from the group consisting of ascorbate,urate, nitrite, vitamin E, α-tocopherol or nicotinate methylester. Insome embodiments of the invention, the composition comprises asurfactant, for example a polysorbate such as Tween 80. In certainembodiments of the invention, the composition comprises a bufferingagent such as citrate, tris(hydroxymethyl)aminomethane (TRIS) ortartrate.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present invention are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent invention may be made without departing from the spirit thereof,and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a sensor design comprising a tubular capsule that isimplanted under the skin and provides optical sensor in response toanalyte (glucose). FIG. 1B shows a view of this capsule. FIG. 1C showsthe relative size of this capsule. FIG. 1D shows a diagram of shows analternative sensor design, one comprising an amperometric analyte sensorformed from a plurality of planar layered elements.

FIG. 2 shows a bar graph of data presenting dose response (DR) retentionas a function of ebeam radiation dose for non-formulated sensors(control sensors not combined with any radioprotectant compositions),triple dose and formulated sensors at 15 kGy. The triple dose is 3×5kGy. The sensors tested were radiated wet in a solution comprising 50 mMTris-buffer saline. The arrow symbolizes that we can retain +80% of DRafter exposure to 15 kGy for formulated sensors.

FIG. 3 shows a plot of phase and intensity data obtained from sensorsafter exposure to 15 kGy of radiation. The dose response is 1.7 afterradiation compared to 2.1 before i.e. a retention of 81%.

FIG. 4 shows a graph of data on DR retained for irradiated sensors as afunction of Ascorbate concentration used for formulation. Too low or toohigh concentrations of Ascorbate used both yield low retained DR whereasthe 20 mM to 100 mM concentration range yields good protection.

FIG. 5 shows a graph of data on DR retained for irradiated sensors as afunction of Acetaminophen (=paracetamol, hence abbreviated PAM)concentration used for formulation. It is seen that using lowconcentrations of Acetaminophen yields low retained DR whereas the useof concentrations above 10 mM yields good protection. Further it isshown that adding Ascorbate to the excipients in most cases providesbetter protective effects.

FIG. 6 shows a graph of data on DR retained for irradiated sensors as afunction of Acetaminophen concentration used for formulation.

FIG. 7 shows a graph of data of DR retained for irradiated sensors as afunction of Acetaminophen concentration used for formulation. Allsensors have contained 100 mM Sucrose and variation of additions ofAscorbate and Mannose are also shown.

FIG. 8 shows a graph of data of DR retained for irradiated sensors as afunction of Ascorbate concentration used for formulation. All sensorshave contained 500 mM Sucrose and variation of additions ofAcetaminophen (PAM) and Mannose are also shown.

FIG. 9 shows a bar graph of data presenting the absolute DR for bothradiated and non-radiated sensor as a function of formulating thesensors with Acetaminophen and Ascorbic acid/ascorbate.

FIG. 10 shows a bar graph of data presenting the absolute DR for bothradiated and non-radiated sensor as a function of formulating thesensors with Acetaminophen, Ascorbic acid, Mannose and 500 mM Sucrose.The overall result is illustrated in FIG. 11.

FIG. 11 shows a graph of data showing sensor response after usingTris/Citrate saline buffer+excipients. Sensors show good retention ofDR.

FIG. 12 shows a graph of data presenting a direct comparison of e-beamedand non e-beamed sensors.

FIG. 13 shows a graph of data obtained from a native sensor tested afterstorage in PBS pH=5.5. The sensor itself has no problem with the PBSbuffer.

FIG. 14 shows a graph of data obtained from a sensor with excipientsadded (500 mM sucrose, 20 mM Acetaminophen and 50 mM Ascorbate) in PBSbuffer during e-beam irradiation.

FIG. 15 shows a graph of data obtained from a sensor with excipientsadded (500 mM sucrose, 20 mM Acetaminophen and 50 mM Ascorbate) in PBSbuffer.

FIG. 16 shows a bar graph of data on retained DR for using differentbuffer concentrations.

FIG. 17 shows a graph of data resulting from sensors using citrate onlyduring e-beam irradiation.

FIG. 18 shows a graph of data resulting from sensors using citrate andexcipients during e-beam irradiation.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

A number of terms are defined below.

The term “sensor” for example in “analyte sensor,” is used in itsordinary sense, including, without limitation, means used to detect acompound such as an analyte. A “sensor system” includes, for example,elements, structures and architectures (e.g. specific 3-dimensionalconstellations of elements) designed to facilitate sensor use andfunction. Sensor systems can include, for example, compositions such asthose having selected material properties, as well as electroniccomponents such as elements and devices used in signal detection (e.g.optical detectors, current detectors, monitors, processors and thelike). The term “sensing complex” as used herein refers to the elementsof a sensor that interact with and generate a signal indicative of, aparticular analyte (e.g. glucose and the like). The term “matrix” isused herein according to its art-accepted meaning of something within orfrom which something else is found, develops, and/or takes form. Whiletypical embodiments of the invention pertain to glucose sensors used inthe management of diabetes, the systems, methods and materials disclosedherein can be adapted for use with a wide variety of medical devicesknown in the art.

In the management of diabetes, the regular measurement of glucose in theblood is essential in order to ensure correct insulin dosing.Furthermore, it has been demonstrated that in the long term care of thediabetic patient better control of the blood glucose levels can delay,if not prevent, the onset of retinopathy, circulatory problems and otherdegenerative diseases often associated with diabetes. Thus, there is aneed for reliable and accurate self-monitoring of blood glucose levelsby diabetic patients. Typically, blood glucose is monitored by diabeticpatients with the use of commercially available colorimetric test stripsor electrochemical biosensors (e.g. enzyme electrodes), both of whichrequire the regular use of a lancet-type instrument to withdraw asuitable amount of blood each time a measurement is made. On average,the majority of diabetic patients would use such instruments to take ameasurement of blood glucose twice a day. However, the U.S. NationalInstitute of Health has recommended that blood glucose testing should becarried out at least four times a day, a recommendation that has beenendorsed by the American Diabetes Association. This increase in thefrequency of blood glucose testing imposes a considerable burden on thediabetic patient, both in financial terms and in terms of pain anddiscomfort, particularly in the long-term diabetic who has to makeregular use of a lancet to draw blood from the fingertips. Thus, thereis clearly a need for a better long-term glucose monitoring system thatdoes not involve drawing blood from the patient.

There have been a number of proposals for glucose measurement techniquesthat do not require blood to be withdrawn from the patient. One methodfor assaying glucose via competitive binding uses a proximity-basedsignal generating/modulating moiety pair which is typically an energytransfer donor acceptor pair (comprising an energy donor moiety and anenergy acceptor moiety). The energy donor moiety is photoluminescent(usually fluorescent). In such methods, an energy transferdonor-acceptor pair is brought into contact with the sample (such assubcutaneous fluid) to be analyzed. The sample is then illuminated andthe resultant emission detected. Either the energy donor moiety or theenergy acceptor moiety of the donor-acceptor pair is bound to a receptorcarrier (for example a carbohydrate binding molecule), while the otherpart of the donor acceptor pair (bound to a ligand carrier, for examplea carbohydrate analogue) and any analyte (for example carbohydrate)present compete for binding sites on the receptor carrier. Energytransfer occurs between the donors and the acceptors when they arebrought together. An example of donor-acceptor energy transfer isfluorescence resonance energy transfer (Förster resonance energytransfer, FRET), which is non-radiative transfer of the excited-stateenergy from the initially excited donor (D) to an acceptor (A). Energytransfer produces a detectable lifetime change (reduction) of thefluorescence of the energy donor moiety. Also, a proportion of thefluorescent signal emitted by the energy donor moiety is quenched. Thelifetime change is reduced or even eliminated by the competitive bindingof the analyte. Thus, by measuring the apparent luminescence lifetime,for example, by phase-modulation fluorometry or time resolvedfluorometry (see Lakowicz, Principles of Fluorescence Spectroscopy,Plenum Press, 1983, Chapter 3), the amount of analyte in the sample canbe determined. The intensity decay time and phase angles of the donorare expected to increase with increasing analyte concentration. Animportant characteristic of FRET is that it occurs over distancescomparable to the dimensions of biological macromolecules. The distanceat which FRET is 50% efficient, called the Förster distance, istypically in the range of 20-60 Å. Förster distances ranging from 20 to90 A are convenient for competitive binding studies. See, e.g. U.S. Pat.No. 6,232,120 and U.S. Patent Application Publication Nos. 20080188723,20090221891, 20090187084 and 20090131773.

WO 91/09312 describes a subcutaneous method and device that employs anaffinity assay based on glucose (incorporating an energy transfer donoracceptor pair) that is interrogated remotely by optical means.WO97/19188, WO 00/02048, WO 03/006992 and WO 02/30275 each describeglucose sensing by energy transfer, which produce an optical signal thatcan be read remotely. The systems discussed above rely on the plantlectin Concanavalin A (Con A) as the carbohydrate binding molecule. WO06/061207 proposes that animal lectins such as mannose binding lectin(MBL) could be used instead. Previously disclosed carbohydrate analogues(e.g. those of U.S. Pat. No. 6,232,130) have comprised globular proteinsto which carbohydrate and energy donor or energy acceptor moieties areconjugated. Carbohydrate polymers (e.g. optionally derivatized dextranand mannan) have also been used as carbohydrate analogues. In WO06/061207 the use of periodate cleavage to allow binding of dextran toMBL at physiological calcium concentrations is disclosed. The assaycomponents in such systems are typically retained by a retainingmaterial. This may for example be a shell of biodegradable polymericmaterial, as described in WO 2005/110207.

Before implantable medical devices such as glucose sensors areintroduced into the body, they must be sterilized. However, thematerials of such devices, for example the assay components in sensors,can be sensitive to damage during sterilization. Heat sterilizationcauses denaturation of protein (lectin and/or carbohydrate analogue).Gas sterilization is difficult to use for wet devices such as thesensor. In view of this, the sterilization of medical devices byexposure to radiation is a common practice. Types of radiation which maybe used in sterilization include gamma radiation and electron beamradiation. Electron beam radiation is easier to control than gammaradiation. However, electron beam radiation can lead to loss of proteinactivity and bleaching of dyes (e.g. a donor fluorophore and/or aacceptor dye). These effects can lead to loss of sensor activity.

Embodiments of the invention provide methods and materials that can beused to protect medical devices such as implantable glucose sensors fromunwanted effects of radiation sterilization. The invention disclosedherein has a number of embodiments. Typical embodiments of the inventioncomprise methods for inhibiting damage to a medical device (e.g. asaccharide sensor) that can result from a radiation sterilizationprocess by combining the medical device with an aqueous radioprotectantformulation during the sterilization process. In the context ofembodiments of the invention as disclosed herein, because electron beamand gamma irradiation are fundamentally the same process, the protectionprovided by the methods and materials of the invention will be the samefor these forms of irradiation. Gamma rays release secondary electronsfrom the materials around the item and hence create a cascade ofelectrons much like the e-beam. For this reason, gamma irradiation issuitable for sensors comprising one or more metal elements because metalis a good provider of secondary electrons. In some embodiments of theinvention, the radiation sterilization process comprises electron beamirradiation. In some embodiments of the invention, the radiationsterilization process comprises gamma ray irradiation.

While the medical devices can be exposed to radiation supplied inmultiple doses (e.g. 3×5 kGy for a total dose of 15 kGy), in typicalembodiments of the instant invention, radiation is supplied in a singledose (e.g. 1×15 kGy for a total dose of 15 kGy). As disclosed herein(see, e.g. FIG. 2), supplying a sterilizing amount radiation in a singledose gives better radiation protection than supplying the same amount ofradiation in multiple doses (dividing the radiation into a triple doseresulted in sensors having worse signal retention). Optionally, thetotal dose of radiation is not more than 35 kGy, and typically is in therange of. 10-20 kGy). In certain embodiments the total dose is 15 kGy±2kGy. Gy (J/kg) is the SI unit of dose i.e. the amount of energy absorbedper unit mass. Following radiation exposure, sensor function parameterscan be evaluated such as the sensor Dose Response (DR relative to 0 kGyDR) as well as the absolute DR (measured in degrees phase shift from 40mg/dL glucose to 400 mg/dL glucose). In certain embodiments of theinvention, an aqueous radiation protecting formulation functions toprotect a glucose sensor from radiation damage so that the glucosesensor retains at least 50, 60 or 70% of its dose response (DR) toglucose following irradiation of the sensor (as compared to the DR of acontrol sensor that received no irradiation).

In some embodiments of the invention, the saccharide sensor comprises aboronic acid derivative such as those disclosed in U.S. Pat. Nos.5,777,060, 6,002,954 and 6,766,183, the contents of which areincorporated herein by reference. In other embodiments of the invention,the saccharide sensor comprises a saccharide binding polypeptide. Incertain embodiments of the invention the saccharide sensor comprises alectin. Optionally the lectin is a C-type (calcium dependent) lectin. Insome embodiments, the lectin is a vertebrate lectin, for example amammalian lectin such as a human or humanized lectin. However, it mayalternatively be a plant lectin, a bird lectin, a fish lectin or aninvertebrate lectin such as an insect lectin. In certain embodiments,the lectin is in multimeric form. Multimeric lectins may be derived fromthe human or animal body. Alternatively, the lectin may be in monomericform. Monomeric lectins may be formed by recombinant methods or bydisrupting the binding between sub-units in a natural multimeric lectinderived from the human or animal body. Examples of this are described inU.S. Pat. No. 6,232,130. Saccharide sensors useful in embodiments of theinvention are also disclosed in U.S. Patent Publication No.2008/0188723, the contents of which are incorporated by reference.

In certain embodiments of the invention, the saccharide sensing elementin a saccharide sensor comprises a lectin. Optionally, the lectin ismannose binding lectin, conglutinin or collectin-43 (e.g. bovine CL-43)(all serum collecting) or a pulmonary surfactant protein (lungcollectins). Mannose binding lectin (also called mannan binding lectinor mannan binding protein, MBL, MBP), for example human MBL, has provedparticularly interesting. MBL is a collagen-like defense molecule whichcomprises several (typically 3 to 4 (MALDI-MS), though distributions of1 to 6 are likely to occur (SDS-PAGE)) sub-units in a “bouquet”arrangement, each composed of three identical polypeptides. Eachsub-unit has a molecular weight of around 75 kDa, and can be optionallycomplexed with one or more MBL associated serine proteases (MASPs). Eachpolypeptide contains a CRD. Thus, each sub-unit presents threecarbohydrate binding sites. Trimeric MBL and tetrameric MBL (which arethe major forms present in human serum, Teillet et al., Journal ofImmunology, 2005, page 2870-2877) present nine and twelve carbohydratebinding sites respectively. In typical embodiments of the invention, thelectin comprises polypeptides of Homo sapiens mannose-binding protein Cprecursor (NCBI Reference Sequence: NP_(—)000233.1). Serum MBL is madeof 3-4 subunits of 3 polypeptides each. The sequence of NCBI ReferenceSequence: NP_(—)000233.1 is between 27 kDa and 30 kDa giving the entireMBL protein a Mw typically of 270 kDa to 300 kDa.

Alternatively, the lectin may be a pulmonary surfactant protein selectedfrom SP-A and SP-D. These proteins are similar to MBL. They arewater-soluble collecting which act as calcium dependent carbohydratebinding proteins in innate host-defense functions. SP-D also bindslipids. SP-A has a “bouquet” structure similar to that of MBL(Kilpatrick D C (2000) Handbook of Animal Lectins, p. 37). SP-D has atetrameric “X” structure with CRDs at each end of the “X”. Othersuitable animal lectins are known in the art such as PC-lectin CTL-1,Keratinocyte membrane lectins, CD94, P35 (synonym: human L-ficolin, agroup of lectins), ERGIC-53 (synonym: MR60), HIP/PAP, CLECSF8, DCL(group of lectins), and the GLUT family proteins, especially GLUT1,GLUT4 and GLUT11. Further suitable animal lectins are set out inAppendices A, B and C of “Handbook of Animal Lectins: Properties andBiomedical Applications”, David C. Kilpatrick, Wiley 2000.

In common embodiments of the invention, the saccharide sensor comprisesa saccharide binding polypeptide having a carbohydrate recognitiondomain and the aqueous radioprotectant formulation comprises asaccharide selected for its ability to bind the saccharide bindingpolypeptide. In certain embodiments of the invention, the saccharidesensor comprises one or more fluorophores (e.g. a donor and/or areference fluorophore); and the aqueous radioprotectant formulationcomprises a fluorophore quenching compound selected for its ability toquench the fluorophore(s). Optionally, the sensor comprises at least oneof protein/polypeptide, at least one energy donor, and/or at least oneenergy acceptor and this sensor is combined with at least one protectivesubstance. In some embodiments the sensor comprises a protein, afluorescent dye, dextran and a polymeric material. In illustrativeembodiments of the invention, the sensor is a glucose sensor and thesaccharide binding polypeptide comprises a mannan binding lectin, aconcanavalin A, a glucose oxidase, or a glucose-galactose bindingprotein (see, e.g. U.S. Pat. No. 6,232,130; U.S. Patent Publication No.2008/0188723; Jensen et al., Langmuir. 2012 Jul. 31; 28(30):11106-14.Epub 2012; Paek et al., Biosens Bioelectron. 2012 and Judge et al.,Diabetes Technol Ther. 2011 March; 13(3):309-17, 2011, the contents ofwhich are incorporated by reference).

As discussed below, a number of compounds are useful in theradioprotectant formulations disclosed herein. In certain embodiments ofthe invention, the aqueous radioprotectant formulation comprises a sugarsuch as glucose, mannose, fructose, melizitose, N-acetyl-D-glucosamine,sucrose or trehalose. In some embodiments, the aqueous radioprotectantformulation comprises an antioxidant selected from the group consistingof ascorbate, urate, nitrite, vitamin E, α-tocopherol or nicotinatemethylester. In certain embodiment of the invention, the aqueousradioprotectant formulation comprises a buffering agent, for example,one selected from the group consisting of citrate,tris(hydroxymethyl)aminomethane (TRIS) and tartrate.

In typical methods of the invention, the sterilization process isperformed under conditions selected to protect the functional integrityof the sterilized sensor. For example, in typical embodiments of theinvention, the sterilization process is performed during or aftercooling the device. In illustrative embodiments, the sterilizationprocess is performed below a certain temperature or within a particularrange of temperatures, for example below 10° C. or below 5° C. or at atemperature between 0 and 5° C., or between 0 and 10° C. In someembodiments of the invention, the sterilization process is performedunder oxygen free conditions (e.g. when a formulation does not comprisean oxidizing compound). Optionally, the process is performed on a sensorwithin and aqueous formulation that has been de-aerated with argon gas,nitrogen gas, or the like. In some embodiments of the invention, thesterilization process is performed using a formulation having a pH below7, below 6, or below 5 etc. In some embodiments of the invention, thesterilization process is performed under conditions selected so that thesaccharide binds the saccharide binding polypeptide and/or thefluorophore quenching composition quenches the fluorophore so as toinhibit damage to the saccharide sensor that can result from theradiation sterilization process. Some methodological embodiments of theinvention comprise further steps, for example those where an irradiatedsensor composition comprising the aqueous radiation protectingformulation is dialyzed to alter the concentrations of one or morecomponents in the formulation.

Another embodiment of the invention is a composition of mattercomprising a saccharide sensor and a fluorophore. The saccharide sensingelement of the saccharide sensor can comprise a boronic acid derivative,a molecular imprinted polymer or a polypeptide. In such compositions,the saccharide sensor is combined with a fluorophore quenching compound.One illustrative embodiment of the invention is a composition of mattercomprising a saccharide sensor that includes a saccharide bindingpolypeptide having a carbohydrate recognition domain; and a fluorophore.In such compositions, the saccharide sensor is combined with an aqueousradioprotectant formulation comprising a saccharide, wherein thesaccharide binds to the carbohydrate recognition domain. Optionally insuch compositions, the saccharide sensor is also combined with afluorophore quenching compound.

A number of compounds can be combined with the saccharide sensorsdisclosed herein to form the radioprotectant compositions of theinvention. In typical embodiments of the invention, the compositioncomprises a saccharide selected from the group consisting of glucose,mannose, fructose, melizitose, N-acetyl-D-glucosamine, GluNac, sucroseor trehalose. In certain embodiment of the invention, the compositioncomprises a fluorophore quenching compound, for example, acetaminophen.In some embodiments of the invention, the composition comprises anantioxidant compound is selected from the group consisting of ascorbate,urate, nitrite, vitamin E, α-tocopherol or nicotinate methylester. Insome embodiments of the invention, the composition comprises asurfactant, for example a polysorbate such as Tween 80. In certainembodiments of the invention, the composition comprises a bufferingagent such as citrate, tris(hydroxymethyl)aminomethane (TRIS) ortartrate. Optionally the composition is formed to have a pH of 7 orbelow, 6 or below, or 5 or below.

Specific compounds are observed to provide saccharide sensors (e.g.those shown in FIGS. 1A-1C) with high levels of protection againstradiation damage when present in aqueous radioprotectant formulations ina particular concentration range. For example, in certain embodiments ofthe invention, the radiation protecting formulation comprisesacetaminophen in a concentration of at least 1 mM to 50 mM (e.g. atleast 10 mM, at least 20 mM, at least 30 mM, at least 40 mM etc.).Optionally, the radiation protecting formulation comprises acetaminophenin a concentration of 20 mM±10 mM (and typically ±5 mM). In certainembodiments of the invention, the radiation protecting formulationcomprises sucrose in a concentration of at least 10 mM to 1000 mM (e.g.at least 100 mM, at least 200 mM, at least 300 mM, at least 400 mMetc.). Optionally, the radiation protecting formulation comprisessucrose in a concentration of 500 mM±200 mM (and typically ±100 mM). Incertain embodiments of the invention, the radiation protectingformulation comprises mannose in a concentration of at least 1 mM to 100mM (e.g. at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mMetc.). Optionally, the radiation protecting formulation comprisesmannose in a concentration of 50 mM±20 mM (and typically ±10 mM). Incertain embodiments of the invention, the radiation protectingformulation comprises ascorbate in a concentration of at least 1 mM to100 mM (e.g. at least 10 mM, at least 20 mM, at least 30 mM, at least 40mM etc.). In certain embodiments of the invention, the radiationprotecting formulation comprises ascorbate in a concentration of notmore than 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mMor 100 mM. Optionally, the radiation protecting formulation comprisesascorbate in a concentration of 50 mM±20 mM (and typically ±10 mM). Incertain embodiments of the invention, the radiation protectingformulation comprises Tris in a concentration of at least 1 mM to 10 mM(e.g. at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM etc.).Optionally, the radiation protecting formulation comprises Tris in aconcentration of 5 mM±2 mM (and typically ±1 mM). In certain embodimentsof the invention, the radiation protecting formulation comprises citratein a concentration of at least 5 mM to 100 mM (e.g. at least 10 mM, atleast 20 mM, at least 30 mM, at least 40 mM etc.). Optionally, theradiation protecting formulation comprises citrate in a concentration of10 mM±2 mM (and typically ±1 mM).

As shown by the working embodiments disclosed herein, one or more ofthese compounds is typically combined with another of these compounds inthe radiation protecting formulations of the invention. For example,certain formulations of the invention will comprise sucrose combinedwith acetaminophen and/or ascorbate and/or Tris and/or citrate.Similarly, certain formulations of the invention will compriseacetaminophen combined with sucrose and/or ascorbate and/or Tris and/orcitrate. Similarly, certain formulations of the invention will compriseascorbate combined with sucrose and/or acetaminophen and/or Tris and/orcitrate. Similarly, certain formulations of the invention will comprisecitrate combined with sucrose and/or acetaminophen and/or Tris and/orascorbate. The formulations can comprise additional compositions such asone or more amino acids or pharmaceutically acceptable salts, forexample those disclosed in Remington: The Science and Practice ofPharmacy, University of the Sciences in Philadelphia (Ed), 21^(st)Edition (2005). As the sensor is to be used in the body, in typicalembodiments, the excipients are commonly acceptable for use in the body.

As noted above, embodiments of the invention disclosed herein providemethods and materials useful in sterilization procedures for medicaldevices such as glucose sensors. While glucose sensors are the commonembodiment discussed herein, embodiments of the invention describedherein can be adapted and implemented with a wide variety of medicaldevices. As discussed in detail below, typical sensors that benefit fromthe methods and materials of the invention include, for example, thosehaving sensing complexes that generate an optical signal that can becorrelated with the concentration of an analyte such as glucose. Anumber of these sensors are disclosed, for example in U.S. PatentApplication Publication Nos. 20080188723, 20090221891, 20090187084 and20090131773, the contents of each of which are incorporated herein byreference. Embodiments of the invention described herein can also beadapted and implemented with amperometric sensor structures, for examplethose disclosed in U.S. Patent Application Publication Nos. 20070227907,20100025238, 20110319734 and 20110152654, the contents of each of whichare incorporated herein by reference.

The compositions used in embodiments of the invention exhibit asurprising degree of flexibility and versatility, characteristics whichallow them to be adapted for use in a wide variety of sensor structures.In some embodiments of the invention, one or more sensor elements cancomprise a structure formed from a polymeric composition through whichwater and other compounds such as analytes (e.g. glucose) can diffuse.Illustrative polymeric compositions are disclosed in U.S. PatentPublication No. 20090221891 and include, for example, material (e.g. onethat is biodegradable) comprising a polymer having hydrophobic andhydrophilic units. Specific polymers can be selected depending upon adesired application. For example, for mobility of glucose, a materialcan be selected to have a molecular weight cut-off limit of no more than25000 Da or no more than 10000 Da. Components disposed within suchpolymeric materials (e.g. sensing complexes) can be of high molecularweight, for example proteins or polymers, in order to prevent their lossfrom the sensor by diffusion through the polymeric materials. In anillustrative embodiment, hydrophilic units of a polymeric materialcomprise an ester of polyethylene glycol (PEG) and a diacid, and themolecular weight cut-off limit is affected by the PEG chain length, themolecular weight of the polymer and the weight fraction of thehydrophilic units. The longer the PEG chains, the higher the molecularweight cut-off limit, the higher the molecular weight of the polymer,the lower the molecular weight cut-off limit, and the lower the weightfraction of the hydrophilic units, the lower the molecular weightcut-off limit.

Sensor components can be selected to have properties that facilitatetheir storage and or sterilization. In some embodiments of theinvention, all components of the sensor are selected for an ability toretain sensor function following a sterilization procedure (e.g. e-beamsterilization). In some embodiments of the invention, all components ofthe sensor are selected for an ability to retain sensor functionfollowing a drying procedure (e.g. lyophilization).

In illustrative embodiments of the invention, the sensor comprises acylindrical/tubular architecture and has a diameter of less than 1 mm,0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm or 0.2 mm.Illustrative sensors of this type are shown in FIG. 1. In certainexamples, the sensor has a diameter of about 0.5 mm or about 0.25 mm. Insome embodiments, the body of sensor is formed from a polymericmaterial. Optionally, the sensor is in the form of a fiber. In someembodiments of the invention, the internal matrix of a cylindricalsensor comprises one or more cavities or voids, for example aencapsulated longitudinal cavity.

Optionally the sensing complex produces an optical signal that can becorrelated with an analyte of interest, for example, glucose. A sensingcomplex (e.g. one comprising a binding assay) generating the opticalsignal should typically be reversible such that a continuous monitoringof fluctuating levels of analyte can be achieved. Optionally, thedetectable or measurable optical signal is generated using a proximitybased signal generating/modulating moiety pair so that a signal isgenerated or modulated when a first member of the pair is brought intoclose proximity with a second member of the pair. In one illustrativeembodiment, the analyte binding agent (e.g. a lectin such as mannosebinding lectin as disclosed in WO 2006/061207) is labelled with one of aproximity based signal generating/modulating moiety pair and the analyteanalogue is labelled with the other of the proximity based signalgenerating/modulating moiety pair, and there is a detectable differencein signal when the analyte analogue and analyte binding agent form thecomplex and when the analyte analogue is displaced by the analyte fromthe complex. Typically, the proximity based signal generating/modulatingmoiety pair is an energy donor moiety and energy acceptor moiety pair.Energy donor moieties and energy acceptor moieties are also referred toas donor and acceptor chromophores (or light absorbing materials)respectively. An energy acceptor which does not emit fluorescence isreferred to as a quenching moiety. In such embodiments, a lectin can belabelled with one of an energy donor and energy acceptor moiety pair andthe analyte analogue is labelled with the other of the energy donor andenergy acceptor moiety pair. The detectable difference in signalcorresponds to a detectable difference in energy transfer from theenergy donor moiety to the energy acceptor moiety. Optionally, theanalyte analogue bears the energy acceptor moiety and the analytebinding agent bears the energy donor moiety. In certain embodiments ofthe invention, the sensor of the invention incorporates an assay whichgenerates an optical readout using the technique of fluorescenceresonance energy transfer (FRET).

In one illustrative embodiment of the sensors discussed in the paragraphabove, the variants of the competitive binding assay each comprise: ananalyte binding agent labelled with a first light-absorbing material; amacromolecule labelled with a second light-absorbing material andcomprising at least one analyte analogue moiety; wherein the analytebinding agent binds at least one analyte analogue moiety of themacromolecule to form a complex from which said macromolecule isdisplaceable by said analyte, and wherein said complex is able to absorblight energy and said absorbed light energy is able to benon-radiatively transferred between one of the light-absorbing materialsand the other of the light-absorbing materials with a consequentmeasurable change in a fluorescence property of said light absorbingmaterials when present in said complex as compared to their saidfluorescence property when said macromolecule is displaced by saidanalyte from said complex, and wherein the different variants of theassay are distinguished by the number of analyte analogue moietiespresent in the macromolecule. Such sensors are disclosed, for example inU.S. Patent Application Publication Nos. 20080188723, 20090221891,20090187084 and 20090131773, the contents of each of which areincorporated herein by reference.

In other embodiments of the invention, the sensor comprises planarlayered elements and, for example comprises a conductive layer includingan electrode, an analyte sensing layer disposed over the conductivelayer (e.g. one comprising glucose oxidase); and an analyte modulatinglayer disposed over the analyte sensing layer. In certain embodiments ofthe invention, the sensor electrode is disposed within a housing (e.g. alumen of a catheter). The sensor embodiment shown in FIG. 1D is aamperometric sensor 100 having a plurality of layered elements includinga base layer 102, a conductive layer 104 which is disposed on and/orcombined with the base layer 102. Typically the conductive layer 104comprises one or more electrodes. An analyte sensing layer 110(typically comprising an enzyme such as glucose oxidase) is disposed onone or more of the exposed electrodes of the conductive layer 104. Aprotein layer 116 disposed upon the analyte sensing layer 110. Ananalyte modulating layer 112 is disposed above the analyte sensing layer110 to regulate analyte (e.g. glucose) access with the analyte sensinglayer 110. An adhesion promoter layer 114 is disposed between layerssuch as the analyte modulating layer 112 and the analyte sensing layer110 as shown in FIG. 1D in order to facilitate their contact and/oradhesion. This embodiment also comprises a cover layer 106 such as apolymer coating can be disposed on portions of the sensor 100. Apertures108 can be formed in one or more layers of such sensors. Amperometricglucose sensors having this type of design are disclosed, for exampleare disclosed, for example, in U.S. Patent Application Publication Nos.20070227907, 20100025238, 20110319734 and 20110152654, the contents ofeach of which are incorporated herein by reference.

Embodiments of the invention can be used with sensors having a varietyof configurations and/or sensing complexes. In certain methodologicalembodiments of the invention, the sensor comprises a cylindricalpolymeric material having a diameter of less than 1 mm, less than 0.5 mmor less than 0.25 mm, the internal matrix comprises an encapsulatedlongitudinal cavity, and the sensing complex comprises a carbohydratebinding lectin (e.g. mannose binding lectin which binds glucose) coupledto a fluorophore pair. In other methodological embodiments of theinvention, the sensor comprises an electrode coated with glucose oxidaseand a glucose limiting membrane disposed over the glucose oxidase,wherein the glucose limiting membrane modulates the diffusion of glucosetherethrough.

Various publication citations are referenced throughout thespecification. The disclosures of all citations in the specification areexpressly incorporated herein by reference. All numbers recited in thespecification and associated claims that refer to values that can benumerically characterized can be modified by the term “about”.

EXAMPLES Example 1 Illustrative Methods and Materials for Use withEmbodiments of the Invention

Sterilization of medical devices is important and the choice ofsterilization method is based on which methods would be both safe andleast destructive to the medical device. Three methods of sterilizationare commonly used with medical devices. These are heat sterilization,gas sterilization and radiation sterilization. Heat sterilization can beproblematical for devices that include proteins because the heat candenature the proteins (protein unfolding happens at approx. 60° C.). Gassterilization process can be difficult to use in medical devices thatend up as a wet device because getting a gas into even small amounts ofliquid (and out again) can be difficult. For these reasons radiationsterilization is a method of choice for use with many devices such asthe glucose sensors discussed herein. Moreover, as e-beam is typicallyeasier to control than gamma radiation, e-beam radiation is used in theillustrative examples disclosed herein. As noted below, e-beam radiationof protein containing solutions can lead to a loss of protein activityin these sensors. In addition, e-beam radiation of dyes can lead tobleaching of the dyes. Both these effects can contribute to losses insensor activity.

In aqueous solutions, the radiolysis of water can initiate oxidationreactions of compounds dissolved in water. The treatment of aqueoussolutions by electron beam irradiation can decrease the concentration ofcertain compounds, provided that the energy absorbed (dose) issufficient.

During radiolysis (e.g. electron beam; eb) H2O turns into the followingspecies:

OH., eaq, H., H3O+, H2, H2O2

H2O+eb→[0.28]OH.+[0.27]e-(aq)+[0.6]H.+[0.07]H2O2+[0.27]H3O++[0.05]H2

(brackets show the formation of species in μmoles/J)These entities formed by the radiolysis of water initiate many reactionswith compounds present and in literature phenol degradation is oftenused as model compound to study the effect of the radiolysis.

The ionization of the assay components themselves in the solution isminimal compared to the radiolysis of the aqueous solvent since theconcentration of assay is in the range of μM and the concentration ofwater will be approx. 55 M, i.e. the damaging effects of electron beamradiation to the assay origins from attack from water radiolysisproducts. In the optical sensor assay the protein appears in theconcentration of μM i.e. water is present is 10⁷ times the concentrationof protein.

As discussed in detail below, a number of compounds were identified andtested to assess their ability to protect sensors against radiationdamage.

Protection of Polymers

Embodiments of the invention are designed to protect sensors thatcomprise polymers such as PolyActive™. PolyActive™ is a biodegradablepolymeric drug delivery system. PolyActive represents a series ofpoly(ether ester) multiblock copolymers, based on poly(ethylene glycol),PEG, and poly(butylene terephthalate), PBT.

Polymers such as PolyActive™ can be protected against radiation damagesby the presence of α-tocopherol. The α-tocopherol is added to thepolymer by the manufacturer and is an antioxidant (Vitamin E) often usedto protect products against radiation damage. In the PolyActive polymerused in the optical sensor it is expected that the α-tocopherolpredominantly will be in the lipophilic domains of the polymer.

Decoloration of Dyes

Embodiments of the invention are designed to protect sensors thatcomprise dyes such as Alexa Fluor® fluorescent dyes. Decoloration of dyecontaining water, happens when the extensive electron conjugated systemof the dye molecules is destroyed. The presence of radicals in thesolution can initiate this process.

Protein Degradation

Embodiments of the invention are designed to protect sensors thatcomprise proteins such as MBL. Radiation damages to proteins are mostoften initiated by the damage of the disulphide bond RSSR formed by thecysteine residues. Cysteine amino acids are the most affected amino acidby radiation. Radiation damages occur when disulfide bridges break andcarbonyl groups of acidic residues lose their definition thus causingthe proteins to lose their activity.

The MBL protein has cysteine rich N-terminal domains (see, e.g. NCBIReference Sequence: NP_(—)000233.1). The tertiary structure of MBL ismaintained by the RSSR bridges in the N-Terminal and if these are brokenthe structure of the protein and hence the function of the protein islost. Wallis et al., J Biol Chem 274: 3580 (1999) shows a schematic of apolypeptide unit of MBL. In order to protect the protein from radiationdamages one can endeavor to protect the cysteine residues of theN-Terminal and the CRD's.

Protection Against Radiation Damages

Art teaches that the prime species that damages proteins and othermolecules in solution is the OH. (hydroxyl radical) hence this is thespecies to look for during protection. Antioxidants such as ascorbatecan be used to protect proteins from damages by ionizing radiation.Prior art shows that the concentration of ascorbate used to protect theproteins is 0.2 M or higher, most likely due to the need for continuousantioxidant protection.

Antioxidants (e.g. ascorbate) have been described in literature for usein radiation protection of dyes. Vandat et al., Radiation Physics andChemistry 79 (2010) 33-35 reports that electron beam irradiation inducedoxidation leading to decoloration and decomposition of the dye C.I.Direct Black 22. Holton, J. Synchotron Rad. (2009), 16, 133-142 reportsthat ascorbate, nicotinic acid, DNTB, nitrate ion, 1,4-benzoquinone,TEMP and DTT have a protective effect against radiation damage toprotein crystals. Wong et al., Food Chemistry 74 (2001) 75-84 reportsthe effect of L-ascorbic acid (LAA) on oxidative damage to lipid(linoleic acid emulsion) caused by electron beam radiation.

Ascorbate Action

The mechanism of action of protectants is to, for example, scavenge theradicals formed by radiolysis. The ascorbate is capable of reducing thehydroxyl radical. The ascorbate radical will undergo several processese.g. disproportionately to ascorbate and dehydro-ascorbate (DHA). Due tothis possible mode of action (ascorbate radical acting both as oxidizerand reducer) too high a concentration of ascorbate could be damaging tothe chemistry of certain sensor embodiments.

Acetaminophen Action

Acetaminophen is easily oxidized in aqueous solution and hence is ableto reduce radicals in solution. Since this compound also works as afluorescence quencher for the AF594 donor fluorophore and AF700reference fluorophore in a glucose assay system with these components,it appears that acetaminophen protects the dyes from bleaching due toits presence near the lipophilic areas of both the protein and the dyes.

Acetaminophen is more lipophilic than ascorbate and could hence act as alipophilic radical scavenger primarily protecting the vulnerable domains(RSSR bridges and aromatic systems of the dyes) close to lipophilicdomains in the compounds needing protection. This predominant lipophilicprotection from acetaminophen combined with ascorbate's high solubilityin aqueous solution protecting the more hydrophilic domains can be apowerful combination when looking for protection.

Sucrose and Mannose

Polyols like mannitol may be good radical scavenges and hence suchcarbohydrates also could yield some protection against radiation damages(hydrophilic domains). Further sucrose is known to have a stabilizingeffect on the MBL hence this could help to improve the storage stabilityof the assay and mannose would bind to the CRD and create somestabilization effect here. Indeed carbohydrates add protective effectsto the assay.

Buffer System:

Amine containing buffer systems like Tris and HEPES are known to providesome protection to the proteins. Especially they provide protectionagainst tryptophan loss from proteins. We also observe protectiveeffects from Tris buffer. Using Citrate as part of the buffer systemkeeps pH around 6 during storage. Citrate is a tertiary alcohol andalcohols like t-butanol (a tertiary alcohol) and isopropyl alcohol (asecondary alcohol) is known scavengers for radiolysis radicals.

In initial e-beam experiments, sterilization at a 15 kGy dose was usedfor the optical glucose sensor, one that comprises both MBL andfluorophore compositions. The conclusion was further that we wouldcontinue to identify and test excipients first for their individualprotection capability and later take the best from each class and usethem in combination.

The following experiments were all conducted with radiation dose of 15kGy while the sensors were cooled and oxygen free (except when theexcipient was an oxidizing compound). After radiation the performance ofthe sensors was evaluated. The primary parameters evaluated as beingretained was the Dose Response (DR relative to 0 kGy DR) as well as theabsolute DR (measured in degrees phase shift from 40 mg/dL glucose to400 mg/dL glucose) after the 15 kGy radiation dose. Also, sensor signaldrift after radiation was observed but not quantified. The first initialexperiments with sterilizing unformulated sensors (control sensors notcombined with any radioprotectant compositions) yielded the resultsshown in FIG. 2. FIG. 2 shows a graph of data from experiments observinga retained dose response for unformulated sensors as a function ofe-beam doses. The triple dose is 3×5 kGy. The sensors tested wereradiated wet in a solution comprising 50 mM Tris-buffer saline.

A dose of 15 kGy as target for the radiation dose is a reasonable choiceas there is still 50% retention of DR after irradiation of theunformulated fluorescent sensors. In addition the electrochemicalsensors discussed herein are irradiated with 16 kGy if they have a lowbioburden after production (<1.5 cfu). Due to the simplicity of theproduction of the optical sensor we expect this low bioburden to be therule (and not the exception). Hence, a 15 kGy dose of e-beam is expectedto provide sterility.

Tests of Excipients Useful to Protect Fluorescent Sensors from RadiationDamage:

Experiments were conducted on the fluorescent glucose sensor shown inFIGS. 1A-1C, one comprising MBL and fluorophore compounds (see, e.g.U.S. patent application publication 2008/0188723). Excipients used forprotecting the sensor during e-beam sterilization processes wereconsequently chosen to protect MBL and these fluorophores. Dextran wasconsidered to benefit from the protection applied to MBL. The protectiveexcipients were chosen from the following categories:

Known MBL Binding Sugars:

Binding sugars can protect the carbohydrate recognizing domain (CRD) ofthe protein, by keeping the peptide structure in the right conformation.However this is not thermodynamically favored compared to non-bindingsugars. ΔG=ΔH−TΔS. For binding sugars the TΔS contribution is large dueto the binding sugar in the CRD being in an ordered conformation insteadof the random (non-ordered) water structure in the CRD. The bindingsugar will then lower the loss in ΔG less than a low binding sugar dueto entropy effects.

Low-Binding Sugars:

Low-binding sugars can function to provide a more rigid hydrogen-bondingscaffold (compared to water) to support the structure of the proteinduring radiation.

Antioxidants:

Antioxidants are generally used as protective agents against freeradical associated radiation damage. Antioxidants quench radicals byreducing them.

Oxidants:

Oxidants were tested as protective agent for the reduction of thefluorescent dyes. Radicals generated during irradiation could reduce thedyes resulting in bleaching them.

Oxidants could oxidize the dye-radicals formed thus protecting the dyes.Further in this context these compounds were trialed also to show thebenefit of using antioxidants.

Amino Acids:

Amino acids are often used to stabilize pharmaceutical formulations.Both hydrophilic and hydrophobic amino acids were tested.

Surfactants:

Surfactants are often used to stabilize pharmaceutical formulationssince denaturing often happens at phase transitions or boundaries.

Phenyl Compounds:

Phenyl containing compounds may stabilize the fluorescent dyes via a π-πstacking mechanism (and hence the assay).

Bacteriostats:

Bacteriostat compounds tested were phenyl containing compounds.

In the following experiments, two or more excipients were chosen fromeach category and tested individually and in combination with ascorbate.For the best excipients in four categories a larger matrix ofexperiments was trialed.

Results from Screening Round

The list of excipients tested and the concentration of each is shown inTable 1 below.

TABLE 1 A list of tested excipients to protect our sensors duringradiation. All excipients were dialyzed into the sensor prior toirradiation. The sensors formulated with oxidative excipients were notde-aerated prior to radiation all other were de-aerated with Ar. UsedExcipient Excipients Concentration in combi- Type Used range testednations¹⁾ Binding Mannose 1, 2, 5, 10, 20 and 50 mM Yes Sugars Fructose50 mM No Melizitose 20 mM No Low-Binding Sucrose 100, 500 and 1000 mMYes Sugars Trehalose 500 and 1000 mM Yes Antioxidants Ascorbate 5, 50,100 and 250 mM Yes Nitrite 5, 10 and 20 mM Yes Ureate 1 and 5 mM Yesα-Tocopherol  1 mg/mL (4.6 mM) Yes Nicotinate 20 and 50 mM Nomethylester Oxidants H₂O₂ 50 mM Yes N₂O Sat'd (gas bubbled through) NoAmino Acids Lysine 2 mg/mL No Tryptophan 2 mg/mL No Phenylalanine 2mg/mL No Surfactants Synperonic 1 mg/mL No Tween 20 1 mg/mL No Tween 801 mg/mL No “Drugs” Acetaminophen 1, 2, 5, 10 and 20 mM YesAcetylsalicylic 10 mM Yes acid α-Tocopherol  1 mg/mL (4.6 mM) Yes PhenylAcetaminophen 1, 2, 5, 10 and 20 mM Yes Containing Acetylsalicylic 10 mMYes Compounds acid α-Tocopherol  1 mg/mL (4.6 mM) Yes Phenol  1 mg/mL(106 mM) Yes m-Cresol 1 mg/mL (92 mM) Yes Tryptophan 2 mg/mL (98 mM) YesPhenylalanine 2 mg/mL No Nicotinate 20 and 50 mM No methylesterBacteriostats Phenol  1 mg/mL (106 mM) Yes m-Cresol 1 mg/mL (92 mM) YesCombinations +80 Max molarity 1M ¹⁾The combination most often used wastogether with ascorbate.

The excipients listed in Table 1 were evaluated in order to choose whichcompounds should be used for the test of different combination ofexcipient. Test endeavored to identify compounds that individually hadan expected protective property towards a preferred target (e.g. CRD,Dye, General peptide bond or protein and storage stabilizing effects).

Table 2 provides a brief summary of the results of the screening round.In Table 2, an overview of the excipients tested as protective agentsagainst radiation damages during e-beam (15 kGy dose) is provided. Theexcipients are listed according to class of compound. Some of theexcipients are listed in more than one category.

TABLE 2 Retention Range (with Excipient Type Excipients acc. DR) Best InClass Binding Mannose 47%-55% Mannose Sugars Fructose MelizitoseNon-Binding Sucrose 47%-90% Sucrose Sugars Trehalose AntioxidantsAscorbate 28%-80% Ascorbate Nitrite Ureate α-Tocopherol Nicotinatemethylester Oxidants H₂O₂ 38%-58% N₂O N₂O Amino Acids Lysine   44%-95%¹⁾Tryptophan Tryptophan Phenylalanine Surfactants Synperonic 26%-33%Synperonic Tween 20 Tween 80 “Drugs” Acetaminophen 10%-80% AcetaminophenAcetylsalicylic acid α-Tocopherol Phenyl Acetaminophen 10%-80%Acetaminophen Containing Acetylsalicylic acid Compounds α-TocopherolPhenol m-Cresol Tryptophan Phenylalanine Nicotinate methylesterBacteriostats Phenol 0% N/A m-Cresol Combinations +80   50-+80%From Table 2 we chose the following four excipients (all best in theirexcipient class) to be used in combination as follows:

Ascorbate: Used for general protection of the peptide bonds in proteins.In literature mentioned as the best antioxidant and yielding bestprotection of proteins against free radical attack. However inliterature the best protection is obtained with very high concentrationsof ascorbate, most often >200 mM which is at least four times the bestconcentration identified herein. Surprisingly, in tests of the sensorembodiments disclosed herein, it was found that using highconcentrations of ascorbate (e.g. 250 mM) yields poor protection whilelow concentrations of ascorbate (e.g. not more than 100 mM, not morethan 50 mM etc.) yields good protection.

Acetaminophen: This compound is not known to interfere with the proteinin the assay. However it works as a dynamic and reversible quencher ofthe fluorescence from AF594. This means that acetaminophen has an effecton the AF594 and could help to protect the dye from radiation damages,e.g. prevent bleaching.

Mannose: Mannose could protect the carbohydrate recognizing domain (CRD)of the protein, by keeping the peptide structure in the rightconformation.

Sucrose: Sucrose is often used for building a more rigidhydrogen-bonding scaffold (compared to water) to support the structureof the protein during radiation. Also Sucrose could bring some improvedstorage stability to the assay.

The list of combinations with the concentration of each excipient andthe results are shown in Table 3: Table 3 shows 48 variations over thefour chosen excipients that have been tested. The order of thevariations is stochastic.

TABLE 3 Excipient concentration (mM) Dose response Aceta- 0 15 Ascorbateminophen Mannose Sucrose kGy kGy Retained¹⁾ 50 20 5 1.6 1.8 112.5% 50 205 500 1.1 1.6 145.5% *50 20 1 1.8 2.1 116.7% 50 20 1 100 2.2 1.9 86.4%*50 20 1 500 1.5 2.1 140.0% 50 10 5 1.8 1.7 94.4% 50 10 5 100 1.5 1.8120.0% 50 10 5 500 2.0 1.6 80.0% 50 10 1 1.5 1.0 66.7% 50 10 1 100 1.81.4 77.8% 50 10 1 500 1.7 1.0 58.8% 50 10 1.9 1.7 89.5% 50 5 5 0.8 1.7212.5% 50 2 2.0 1.1 55.0% 50 5 2.1 1.7 81.0% 50 5 100 1.9 1.4 73.7% 50 5500 1.7 1.8 105.9% 50 1 1.8 1.7 94.4% 50 1 100 1.7 1.5 88.2% 50 1 5001.8 1.8 100.0% 50 1000 2.3 1.8 78.3% 20 10 2.0 1.7 85.0% 10 20 5 1.6 1.062.5% 10 20 5 100 0.8 0.8 100.0% 10 20 5 500 2.2 1.2 54.5% 10 20 1 1.91.9 100.0% 10 20 1 100 0.9 0.8 88.9% 10 20 1 500 2.1 1.7 81.0% 10 10 51.3 1.5 115.4% 10 10 5 100 1.6 1.7 106.3% 10 10 5 500 1.7 1.6 94.1% 1010 1 1.7 1.5 88.2% 10 10 1 100 1.7 1.0 58.8% 10 10 1 500 2.0 1.9 95.0%10 5 5 1.9 1.3 68.4% 10 2 1.8 1.0 55.6% 10 5 100 1.8 1.6 88.9% 10 1 1.00.0 0.0% 10 1 100 1.8 1.5 83.3% 5 2 1.8 1.2 66.7% 5 1000 2.3 1.8 78.3%20 2.2 1.7 77.3% 10 2.0 1.6 80.0% 5 2.2 1.6 72.7% 2 2.3 1.5 63.0% 1 2.41.3 54.2% 100 2.8 1.7 60.7% 500 1.8 1.9 105.6% ¹⁾Retained DR >100%should not be possible but if the 0 kGy DR is unexpected low retained DRcan become >100% *High absolute DR after radiation

The plot of data shown in FIG. 3 from the SITS system shows a test runof a set of sensors that has had good protection during radiation. FIG.3 shows a plot of phase and intensity data obtained from sensors afterexposure to 15 kGy of radiation. The dose response is 1.7 afterradiation compared to 2.1 before i.e. a retention of 81%. Note the longequilibration time of the sensor after startup. This most likely originsfrom the large concentration of sucrose used in the formulation. As isknown in the art, concentrations of agents in aqueous solutions can beeasily changed via processes such as dialysis.

Excipients Individual Effects

In order to get an overview of the effect of the individual excipientsthe results will be visualized as seen FIG. 4. FIG. 4 shows a graph ofdata on DR retained for irradiated sensors as a function of Ascorbateconcentration used for formulation. Too low or too high concentrationsof Ascorbate used both yield low retained DR whereas the 20 mM to 100 mMconcentration range yields good protection.

FIG. 5 shows a graph of data on DR retained for irradiated sensors as afunction of Acetaminophen (=paracetamol, hence abbreviated PAM)concentration used for formulation. It is seen that using lowconcentrations of Acetaminophen yields low retained DR whereas the useof concentrations above 10 mM yields good protection. Further it isshown that adding Ascorbate to the excipients in most cases gives betterprotection.

FIG. 6 shows data of DR retained for irradiated sensors as a function ofAcetaminophen concentration used for formulation.

FIG. 7 shows data of DR retained for irradiated sensors as a function ofAcetaminophen concentration used for formulation. All sensors havecontained 100 mM Sucrose and variation of additions of Ascorbate andMannose are also shown.

FIG. 8 shows data of DR retained for irradiated sensors as a function ofAscorbate concentration used for formulation. All sensors have contained500 mM Sucrose and variation of additions of Acetaminophen (PAM) andMannose are also shown.

FIG. 9 shows a bar graph of data presenting the absolute DR for bothradiated and non-radiated sensor as a function of formulating thesensors with Acetaminophen and Ascorbic acid.

FIG. 10 shows a bar graph of data presenting the absolute DR for bothradiated and non-radiated sensor as a function of formulating thesensors with Acetaminophen, Ascorbic acid, Mannose and 500 mM Sucrose.An overall result is illustrated in FIG. 11.

FIG. 11 shows a graph of data showing sensor response after usingTris/Citrate saline buffer+excipients. Sensors show good retention ofDR.

FIG. 12 shows a graph of data presenting a direct comparison of e-beamedand non e-beamed sensors.

Buffer Impact on the Sensor Dose Response Retentions after e-Beam

Due to a demand for not degrading the polymer used on the sensor pHlevel needs to be around 6 during wet storage.

PBS Buffer Results

FIG. 13 shows a graph of data obtained from a native sensor tested afterstorage in PBS pH=5.5. The sensor itself has no problem with the PBSbuffer.

FIG. 14 shows a graph of data obtained from a sensor with excipientsadded (500 mM sucrose, 20 mM Acetaminophen and 50 mM Ascorbate) in PBSbuffer during e-beam.

FIG. 15 shows a graph of data obtained from a sensor with excipientsadded (500 mM sucrose, 20 mM Acetaminophen and 50 mM Ascorbate) in PBSbuffer. No dose response and large drift is observed even though thesensors have not been e-beamed.

Alternative Buffers

Alternative clinically acceptable buffers are shown in Table 4.

TABLE 4 List of optional buffers in the desired range together withtheir redox state. Primary “Red-Ox Buffer pK1 pK2 pK3 amine State”Comment Phosphoric 2.15 7.20 12.33 No P = +7 +Excipients Acid DR LossGlycine 2.35 9.78 Yes C = +3 Alanine 2.71 9.10 Yes C = +3 Tartaric Acid3.04 4.37 No C = +3 Citrate 3.13 4.76 6.40 No C = +3 Lactate 3.86 No C =+3 Ascorbic Acid 4.17 11.57 No C = +2 Acetic Acid 4.76 No C = +3 UricAcid 5.83 No Solubility problem Carbonic 6.35 10.33 No C = +4 CO₂pressure acid/ to keep pH Bicarbonate Tris 8.06 Yes

Citrate was found to be superior, and tested in up to 50 mMconcentration. Citrate works OK alone but better if Tris is added:

FIG. 16 shows a bar graph of data on retained DR for using differentbuffer concentrations.

FIG. 17 shows a graph of data resulting from sensors using citrate onlyduring e-beam irradiation.

FIG. 18 shows a graph of data resulting from sensors using citrate andexcipients during e-beam irradiation.

Amines to Protect the Chemistry from e-Beam Damages

In certain embodiments, amines can be included in the formulations (e.g.as a good quencher of radicals). Experimental results have shown thatTris (primary amine) by itself provides protection and that thisprotective effect is improved when excipients are added. Illustrativeamines include urea, creatine, creatin, as well as the 20 naturallyoccurring amino acids.

The data in this Example confirms that the effects of a single excipientas well as the effects of combinations of excipients on glucose sensorDR retention following radiation sterilization are unpredictable. Inthese experiments, categories of agents tested included surfactants,amino acids (hydrophilic/hydrophobic), sugars (binding/non-binding),oxidants, antioxidants, drugs, bacteriostats, and combinations of theseagents. The “best-in-class” excipients appear to include ascorbate,mannose, sucrose (high concentration) and acetaminophen (lowconcentration). The experimental data provides evidence thatcombinations of excipients can protect different specific sites orfunctionalities of a sensor against radiation damages. Ascorbate,mannose, sucrose and acetaminophen in combination provide particularlygood signal retention for sensors. Typical embodiments of the inventioninclude a combination of two to four excipients from each group andusing a combination buffer consisting of 5 mM Tris and/or 10 mM Citratesaline buffer. Some embodiments include sensor storage stabilityenhancing agents such as low-binding sugars (sucrose, trehalose andother polyols)

1. A method of inhibiting damage to a saccharide sensor that can resultfrom a radiation sterilization process, the method comprising combiningthe saccharide sensor with an aqueous radioprotectant formulation duringthe sterilization process, wherein: the saccharide sensor comprises asaccharide binding polypeptide having a carbohydrate recognition domain;the aqueous radioprotectant formulation comprises a saccharide selectedfor its ability to bind the saccharide binding polypeptide; andperforming the sterilization process under conditions selected so thatthe saccharide binds the saccharide binding polypeptide, therebyinhibiting damage to the saccharide sensor that can result from theradiation sterilization process.
 2. The method of claim 1, wherein thesaccharide binding polypeptide comprises mannan binding lectin,concanavalin A or glucose oxidase.
 3. The method of claim 1, wherein thesaccharide comprises glucose, mannose, fructose, melizitose,N-acetyl-D-glucosamine, sucrose or trehalose.
 4. The method of claim 1,wherein the aqueous radioprotectant formulation comprises an antioxidantselected from the group consisting of ascorbate, urate, nitrite, vitaminE, tocopherol or nicotinate methylester.
 5. The method of claim 1,wherein: the saccharide sensor comprises a fluorophore; and the aqueousradioprotectant formulation comprises a fluorophore quenchingcomposition selected for its ability to quench the fluorophore.
 6. Themethod of claim 1, wherein the aqueous radioprotectant formulationcomprises a buffering agent.
 7. The method of claim 6, wherein thebuffering agent is selected from the group consisting of citrate,tris(hydroxymethyl)aminomethane (TRIS) and tartrate.
 8. The method ofclaim 1, wherein the radiation sterilization process comprises a singledose of radiation.
 9. The method of claim 1, wherein the radiationsterilization process comprises electron beam irradiation.
 10. Acomposition of matter comprising: a saccharide sensor comprising: asaccharide binding polypeptide having a carbohydrate recognition domain;and a fluorophore; and an aqueous radioprotectant formulationcomprising: a saccharide, wherein the saccharide is bound to thecarbohydrate recognition domain; and a fluorophore quenchingcomposition.
 11. The composition of claim 10, wherein the fluorophorequenching composition comprises acetaminophen.
 12. The composition ofclaim 11, wherein the aqueous radioprotectant formulation comprisesacetaminophen in a concentration of at least 1 mM to 50 mM.
 13. Thecomposition of claim 10, wherein the aqueous radioprotectant formulationcomprises an antioxidant compound is selected from the group consistingof ascorbate, urate, nitrite, vitamin E, α-tocopherol or nicotinatemethylester.
 14. The composition of claim 13, wherein the aqueousradioprotectant formulation comprises ascorbate in a concentration of atleast 1 mM to 100 mM.
 15. The composition of claim 10, wherein thesaccharide is selected from the group consisting of glucose, mannose,fructose, melizitose, N-acetyl-D-glucosamine, sucrose or trehalose. 16.The composition of claim 15, wherein the aqueous radioprotectantformulation comprises mannose in a concentration of at least 1 mM to 100mM.
 17. The composition of claim 15, wherein the aqueous radioprotectantformulation comprises sucrose in a concentration of at least 10 mM to1000 mM.
 18. The composition of claim 10, wherein the aqueousradioprotectant formulation comprises a buffering agent; and has a pH of6 or below.
 19. The composition of claim 18, wherein the buffering agentis selected from the group consisting of citrate,tris(hydroxymethyl)aminomethane (TRIS) and tartrate.
 20. The compositionof claim 10, wherein the aqueous radioprotectant formulation comprises asurfactant.